Phytochemical Investigation of the Constituents ... - ACS Publications

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Phytochemical Investigation of the Constituents Derived from the Australian Plant Macropidia fuliginosa Robert Brkljača,† Jonathan M. White,‡ and Sylvia Urban*,† †

School of Applied Sciences (Discipline of Chemistry), Health Innovations Research Institute (HIRi), RMIT University, GPO Box 2476 V, Melbourne, Victoria 3001, Australia ‡ School of Chemistry and Bio21 Institute, The University of Melbourne Melbourne, Victoria 3010, Australia S Supporting Information *

ABSTRACT: A phytochemical study of the flowers and bulbs derived from the Australian plant Macropidia f uliginosa, involving hyphenated spectroscopic methodologies (HPLCNMR and HPLC-MS), together with conventional isolation strategies, resulted in the identification of 16 constituents (1, 2, 4−17) representative of six different structural classes. Six new compounds (12−17) were identified from the bulbs of the plant. The isolated compounds were assessed for antimicrobial activity, and compound 8 was found to be more potent against P. aeruginosa than ampicillin.

P

11). The structures of three of these compounds (14, 16, and 17) were defined by single-crystal X-ray diffraction in combination with detailed spectroscopic methods, while the complete 2D NMR spectroscopic characterization of 1, 6, and 11 is reported herein for the first time.

lants commonly known as kangaroo paw comprise a number of species belonging to two genera of the family Hemodoraceae, which are endemic to the southwest corner of Western Australia. There are 11 different species of kangaroo paw, 10 of which belong to the Anigozanthos genus and one to the Macropidia genus.1 Various species of the Anigozanthos genus have been studied and have yielded a range of secondary metabolites including phenylphenalenones, dimeric phenylphenalenones, resveratrol analogues, and oxabenzochrysenones.2−7 By contrast, Macropidia f uliginosa, commonly known as black kangaroo paw, which is classified separately from the other kangaroo paw species, has not been extensively studied. The flowers of M. f uliginosa have been reported to produce the two oxabenzochrysenones hemofluorones A (1) and B (2).8 Initially hemofluorone B was reported to have the structure as depicted by 3,8 but this was subsequently revised to 2.3 Other plants belonging to the family Hemodoraceae, such as Hemodorum simulans and Xiphidium caeruleum, also produce oxabenzochrysenones as well as phenylphenalenones,9,10 which are typical secondary metabolites of many of the species derived from this family. As part of our continuing efforts to study the chemical diversity of Australian plants, we recently conducted phytochemical studies of two different Hemodorum species, one of which is used for the treatment of dysentery.10,11 Motivated by the limited studies that have been conducted on M. f uliginosa, together with our interest in the constituents derived from the Hemodoraceae family of plants, specimens of M. f uliginosa were purchased and subjected to a phytochemical study. Investigation of the flowers and bulbs of M. f uliginosa yielded six new (12−17) and 10 known compounds (1, 2, 4− © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The flowers and bulbs of M. f uliginosa were separately extracted with 3:1 MeOH/CH2Cl2, and the solvent was evaporated under reduced pressure and sequentially solvent-partitioned (triturated) into CH2Cl2- and MeOH-soluble fractions, respectively. The crude CH2Cl2 extract of the flowers was found to be more active than the MeOH crude extract and was selected for phytochemical profiling. While both the CH2Cl2and MeOH-soluble fractions of the bulbs displayed antimicrobial activity, only the CH2Cl2 extract was subjected to an online phytochemical profiling study. This selection was made on the basis of preliminary analytical HPLC and 1H NMR analyses, which indicated the presence of more secondary metabolites in the CH2Cl2 crude extract. The CH2Cl2 crude extract showed better separation of the secondary metabolites present. Both the CH2Cl2 and MeOH crude extracts derived from the bulbs of M. f ulginosa were subjected to an off-line phytochemical investigation. On-line Phytochemical Profiling (HPLC-NMR and HPLC-MS). The CH2Cl2 extracts of the bulbs and flowers were subjected to HPLC-NMR and HPLC-MS chemical profiling with a total of 10 peaks detected (see Supporting Received: February 16, 2015

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DOI: 10.1021/acs.jnatprod.5b00161 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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compounds, of which three (tR = 4.11, 12.35, and 19.18 min) were identified as the known compounds 5−7, respectively, based on a dereplication strategy involving the use of the Dictionary of Natural Products to search the molecular formulas and characteristic UV absorbance maxima in combination with the taxonomy of the plant. The WET1D 1 H NMR spectrum and extracted UV profile of the two compounds eluting at tR = 3.28 min (coeluting peaks) showed close similarity to 5, suggesting these compounds were structural analogues containing a formyl (δH 10.25, s) and a methoxy (δH 4.18, s) moiety. The WET1D NMR spectrum of the compound eluting at tR = 11.31 min suggested an unsubstituted phenylphenalenone; however the extracted UV profile was not in agreement, displaying an absorbance below 400 nm. Phenylphenalenones typically display an absorbance at 450 nm,2 suggesting that the compound eluting at tR = 11.31 min was of a closely related structural class. Off-line Phytochemical Isolation. The off-line isolation and fractionation of the crude extracts derived from the flowers and bulbs of M. fulginosa yielded a total of 16 compounds (1, 2, 4−17). The identities of the four compounds (tR = 3.28, 11.31, and 15.05 min) dereplicated in the on-line chemical profiling approach were subsequently confirmed as 12, 13, 14, and 8, respectively. In total the off-line phytochemical isolation approach resulted in the identification of seven known secondary metabolites from the flowers (1, 2, 4, 8−11) and three from the bulbs (5−7), and their NMR and MS data were in accordance with literature values.2−5,8,15−18 Fuliginosins A (12) and B (13) were isolated as brown oils from the bulbs of M. f uliginosa. Comparison of the 1H NMR spectra of 12 and 13 supported the fact that these compounds were structural analogues and that they were closely related to anigopreissin A (5) except that the resonances for the transphenylvinyl system in anigopreissin A (5) were absent in both 12 and 13. The 1H NMR spectrum of fuliginosin A (12) showed the presence of a formyl moiety (δH 9.91, s) which showed key gHMBCAD NMR correlations to the adjacent aromatic ring (C-5 and C-7, respectively), confirming its attachment to position C-6. Complete analyses of the 1D and 2D NMR data (Table 1) allowed for 12 to be identified as the new benzofuran 3-(3,5-dihydroxyphenyl)-4-hydroxy-2-(4hydroxyphenyl)benzofuran-6-carbaldehyde and given the trivial name fuliginosin A. Inspection of the 1H and HSQCAD NMR spectra of fuliginosin B (13) indicated the presence of two methoxy moieties (δH 3.23, s, (6H); δC 52.9) as well as a characteristic deshielded methine [δH 5.36, s, (1H); δC 103.1]. Key gHMBCAD NMR correlations indicated that these methoxy moieties and the deshielded methine were attached to an aromatic ring. Specifically, the deshielded methine proton showed correlations to the carbons at positions C-5 and C-7 together with correlations to the two methoxy moieties, which confirmed the attachment of this structural unit to C-6. Analyses of the 1D and 2D NMR data (Table 1) allowed for 13 to be identified as the new benzofuran 5-[6-dimethoxymethyl4-hydroxy-2-(4-hydroxyphenyl)benzofuran-3-yl]benzene-1,3diol and given the trivial name fuliginosin B. Fuliginosin B (13) is the dimethyl acetal of fuliginosin A (12), and it was suspected that 13 could be an artifact produced from 12 as a result of the extraction with MeOH. An attempt to convert fuliginosin A (12) to fuliginosin B (13), whereby the former was stored in MeOH as well as in a mixture of 3:1 MeOH/CH2Cl2, showed no conversion after a period of approximately 1 week. This

Information). While the CH2Cl2 crude extract of the bulbs displayed reasonable solubility in MeCN and D2O, the CH2Cl2 crude extract of the flowers did not. This meant that HPLCNMR data could not be obtained for the crude extract derived from the flowers and that dereplication of the constituents present in this extract relied solely on the interpretation of the HPLC-MS and UV spectroscopic data. The process known as dereplication aims to rapidly deduce the chemical classes or compounds present in a crude extract and involves the use of chemical databases such as the Dictionary of Natural Products, MarinLit, or SciFinder. These databases can be utilized to search characteristic UV, MS, and NMR data. HPLC-MS of the CH2Cl2 crude extract of the flowers showed the presence of four compounds, each displaying unique UV absorbance maxima. The molecular formula of the compound eluting at tR = 2.45 min was determined as C27H30O16 [observed m/z at 609.1453 [M − H]− (calcd for C27H29O16: m/z 609.1456)], and the compound displayed UV absorbance maxima above 350 nm. A search conducted in the Dictionary of Natural Products using the molecular formula and consideration of the UV absorbance maxima allowed the tentative identification of the compound eluting at tR = 2.45 min as rutin (4). Similar analyses of the data and literature searches conducted for the compounds eluting at tR = 3.74 and 4.44 min resulted in these constituents being tentatively identified as hemofluorones A (1) and B (2), respectively. The fourth compound eluting at tR = 15.05 min could not be identified, but based on the characteristic UV absorbance at 368 nm it was concluded to be a flavonoid,12,13 a common chemical class of the Hemodoraceae.14 The HPLC-NMR and HPLC-MS of the CH2Cl2 crude extract derived from the bulbs displayed the presence of six B



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Table 1. 1H and 13C NMR Data (500 MHz, DMSO-d6) for Fuliginosins A (12) and B (13) fuliginosin A (12) position 2 3 3a 4 5 6 7 7a 1′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 4-OH 4″-OH 3‴-OH 5‴-OH 2a′-OCH3 2b′-OCH3

δC, mult 152.9, 116.1, 116.1, 152.8, 107.3, 124.0, 106.2, 154.5, 192.6, 120.9, 128.7, 116.0, 158.7, 116.0, 128.7, 133.9, 109.0, 158.5, 102.4, 158.5, 109.0,

C C C C CH C CH C CH C CH CH C CH CH C CH C CH C CH

δH (J in Hz)

gCOSY

fuliginosin B (13) δC,a mult

gHMBCAD

7.07, s

1′, 3a,c 4,c 6, 7

7.61, s

1′, 5, 6, 7a

9.91, s

7, 5

7.35, d (9.0) 6.73, d (9.0)

3″ 2″

2, 4″, 6″ 1″, 4″, 5″

6.73, d (9.0) 7.35, d (9.0)

6″ 5″

1″, 3″, 4″ 2, 2″, 4″

6.22, d (2.0)

3, 3‴, 4‴, 6‴

6.20, dd (2.0, 2.0)

2‴, 3‴, 5‴, 6‴

6.22, d (2.0) NDb NDb NDb NDb

3, 2‴, 4‴, 5‴

149.8, 115.8, 118.3, 154.9, 107.2, ND 100.9, 152.3, 103.1, 121.7, 128.4, 115.9, 158.2, 115.9, 128.4, ND 109.1, 158.6, 102.3, 158.6, 109.1,

C C C Cd CH CH Cd CH C CH CH C CH CH CH C CH C CH

52.9, CH3 52.9, CH3

δH (J in Hz)

gCOSY

gHMBCAD

6.63, s

1′, 3a, 7, 7a

6.98, s

1′, 3a, 4b,c 5

5.36, s

7, 5, 2a′-OCH3, 2b′-OCH3

7.27, d (8.5) 6.69, d (8.5)

3″ 2″

2, 4″ 1″, 4″

6.69, d (8.5) 7.27, d (8.5)

6″ 5″

1″, 4″ 2, 4″

6.20, d (2.0)

4‴

3, 3‴, 4‴, 5‴,c 6‴

6.16, dd (2.0, 2.0)

2‴, 6‴

2‴, 3‴, 5‴, 6 ‴

6.20, d (2.0) NDb NDb NDb NDb 3.23, s 3.23, s

4‴

3, 2‴, 3‴,c 4‴, 5‴

1′ 1′

a

Carbon assignments based on HSQCAD and gHMBCAD NMR experiments. bSignal not detected. cIndicates weak or long-range signal. dSignals interchangeable.

Figure 1. Single-crystal X-ray diffraction structures (ORTEP) of fuliginosone (14), fuliginol (16), and 3-chlorofuliginol (17).

respectively). Inspection of the UV spectrum of 14 showed an absorbance at 323 nm, which is not consistent with a phenylphenalenone skeleton (typically >400 nm).2 In addition the 13C NMR spectrum showed the presence of two diagnostic carbonyl signals (δC 187.7 and 186.9), which also was not in accordance with typical phenylphenalenones.9,10,19 HRESIMS

suggested that fuliginosin B (13) may be a natural product, but the possibility of it being an artifact cannot be excluded. Fuliginosone (14) was isolated as bright yellow needles. The 1 H NMR spectrum showed the presence of eight discrete aromatic/olefinic signals (δH 8.30 (1H), 8.29 (1H), 8.15 (1H), 7.87 (1H), 7.85 (1H), 7.72 (2H), 7.55 (2H), and 7.52 (1H), C



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revealed the molecular formula of 14 to be C18H10O2 (observed m/z at 259.0752 [M + H]+ (calcd for C18H11O2: m/z 259.0759). The presence of 18 carbons, as compared to phenylphenalenones, which typically contain 19 carbons, also supported a deviation to that structure class. It was proposed that 14 contained a “dione” moiety in a five-membered ring, rather than containing a “dione” moiety in a six-membered ring. This would account for the reduction in the number of carbons that is typically observed in the phenylphenalenones. A pendant aromatic ring substituted at C-3 or C-5 was established by analysis of the NMR data. Predicted carbon chemical shifts for the two possible structures were compared using Advanced Chemistry Development (ACD) laboratories and ChemDraw software.20,21 Substitution of the pendant aromatic ring at C-3 showed carbon chemical shits of δC 126 and 138 for C-4 and C5, respectively. The alternative C-5-substituted structure showed carbon chemical shifts of δC 122 and 130 for C-3 and C-4, respectively. Two of the predicted carbon chemicals shifts (δC 138 and 130) differ significantly, and on the basis of a comparison of the observed and calculated carbon chemical shifts, the pendant aromatic ring was placed at C-3. A singlecrystal X-ray diffraction (Figure 1) study of 14 enabled the structure to be unequivocally deduced as 3-phenylacenaphthylene-1,2-dione and given the trivial name fuliginosone. Analyses of the 1D and 2D NMR data (Table 2) also supported the structure of fuliginosone (14).

7.17) at C-3 and, therefore, the aromatic ring at C-2. Thus, the structure of compound 15 was defined as 2-phenyl-1Hphenalen-1-one. A literature review indicated that this compound was obtained as a synthetic analogue;22 however the UV and NMR data provided in that study do not support this structure. All reported phenylphenalenones display a diagnostic UV absorbance between 430 and 450 nm;2,5,9,10,19 however, the product obtained in the synthetic study showed an absorbance at 403 nm.22 Second, several NMR signals were not observed in the synthetic compound reported, in particular the proton signal at δH 7.17 and the carbon signals at δC 149.5 and 113.8.22 Thus, this represents the first isolation and characterization of compound 15, and it was given the trivial name fuliginone. Fuliginol (16) was isolated as a brown solid, and analysis of the UV spectrum revealed an absorption at 463 nm, which was supportive of a phenylphenalenone.2 The 1H and 13C NMR spectra indicated the presence of a methoxy moiety (δH 3.10 (3H); δC 61.5), and the IR spectrum supported the presence of a hydroxy group (3369 cm−1). The molecular formula was obtained via ESIMS and deduced as C20H14O4 (observed m/z at 317.0814 [M − H]− (calcd for C20H13O4: m/z 317.0814). This indicated the presence of two hydroxy groups. Analysis of the gHMBCAD NMR data for H-4 revealed correlations to two deshielded oxygenated tertiary carbons (δC 142.8 and 147.8). The methoxy moiety (δH 3.10) also showed an HMBC correlation to the carbon at δC 142.8, indicating that the methoxy moiety was at C-6. Comparison of the NMR data with those of structurally related phenylphenalenones showed that the C-6 methoxy resonances are deshielded (approximately at δH 4.00) compared to their C-5 counterparts (approximately at δH 3.00) due to the shielding effect of the aromatic ring.19 The assignment of the methoxy group at C-6 allowed for subsequent assignment of the C-5 and C-6 chemical shifts at δC 147.8 and 142.8 ppm, respectively. A single-crystal X-ray diffraction study (Figure 1) provided the unequivocal identification for 16 as 2,5-dihydroxy-6-methoxy-7-phenyl-1Hphenalen-1-one, and it was given the trivial name fuliginol (Table 4). Fuliginol (16) was isolated as a pure compound prior to the crystallization process, and NMR, UV, and IR data were acquired on the single compound. During the process of crystallization and in the single-crystal X-ray diffraction study the presence of a cocrystal occurring with 16 (in a ratio of approximately 1:1) was noted. This cocrystal (Figure 1), a chlorinated derivative of 16, was identified as 3-chloro-2,5dihydroxy-6-methoxy-7-phenyl-1H-phenalen-1-one (17) and given the trivial name 3-chlorofuliginol. It is believed that the CHCl3 used during the IR analysis was responsible for the introduction of the chlorine substituent. Upon completion of the X-ray analysis, the crystals were resuspended in the same batch of CHCl3 in an attempt to convert all of 16 to 17; however after 4 weeks the two compounds remained present in a ratio of approximately 1:1. HPLC purification was attempted on the mixture; however, 16 and 17 were inseparable. Complete 1D and 2D NMR, together with MS analysis, were done on the mixture (Table 4). While fuliginol (16) was isolated from the bulbs of the plant, 3-chlorofuliginol (17) may represent an artifact of the isolation process. The absolute configuration of rutin (4) was established by comparison of the specific rotation with an authentic standard. The absolute configurations of compounds 8 and 11 remain

Table 2. 1H and 13C NMR Data (500 MHz, CDCl3) of Fuliginosone (14) position

a

δC, mult

1 2 2a 3 4 5

187.7, 186.9, 130.1, 136.4, 131.1, 132.6,

C C C C CH CHa

5a 6 7

123.9, C 132.5, CHa 128.0, CH

8 8a 8b 1′ 2′ 3′

122.3, 128.1, 146.9, 139.8, 128.5, 129.7,

4′ 5′

129.2, CH 129.7, CH

6′

128.5, CH

CH C C C CH CH

δH (J in Hz)

gCOSY

gHMBCAD

7.87, d (8.5) 8.30, d (8.5)

5 4

2,b 2a, 3, 5a 2a,b 4,b 5a, 8b, 1′b

8.29, d (8.0) 7.85, ddd (8.0, 7.0, 1.0) 8.15, dd (7.0, 1.0)

7 6, 8

5, 5a, 7,b 8, 8b 5a, 8a

7

1, 6, 7,b 8b

7.55, m 7.72, ddd (8.0, 8.0, 1.5) 7.52, m 7.72, ddd (8.0, 8.0, 1.5) 7.55, m

3′ 2′

3, 6′ 1′, 2′, 4′

6′

3′, 5′ 1′, 4′, 6′

5′

3, 2′

Signals interchangeable. bIndicates weak or long-range correlation.

Fuliginone (15) was isolated as a brown solid. The UV spectrum showed a diagnostic absorbance at 430 nm, confirming a phenylphenalenone-type structure.2 Two spin systems were evident from H-4 to H-6 and from H-7 to H-9 on the basis of gCOSY and gHMBCAD NMR data. An aromatic proton (δH 7.17) and a pendant aromatic ring could be located at either C-2 or C-3, respectively. Key NOE enhancements were observed between H-4 (δH 7.72) and the proton at δH 7.17. This secured the position of the aromatic proton (δH D



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Panama’s disease,28 antifungal activity against Mycosphaerella f ijiensis,29,30 and activities as a nematostatic and nematocidal compound.31 Methyl trans-p-coumarate (9) displays antimelanogenic,32 antiadipogenic,33 anti-inflammatory,34 and antioxidant activities,16 along with α-glucosidase inhibitory effects.16 Methyl cis-p-coumarate (10) has been reported as an antiradical compound.35 Isosalipurposide (11) shows a range of activity including cytotoxicity against CaCo-236 and L929 cells37 and antileishmanial,38 anticarcinogenic,39 and anticancer activities.40 Antimicrobial evaluation of the crude extracts and the isolated compounds was carried out against five bacteria and one fungus. Crude extracts and mixtures were tested at 50, 25, or 1 mg/mL, while pure compounds, together with a commercially available antibiotic and antifungal agent, were tested between 1639 and 5230 μM (Table 5). Compared to the antibiotic ampicillin, the isolated compounds were less active; however compound 8 was more potent against Pseudomonas aeruginosa than ampicillin. In recent years concern has grown around P. aeruginosa with regard to its prevalence and multidrug resistance;41 thus the identification of possible drug leads against this bacteria is important. Thus, on-line phytochemical profiling studies conducted via HPLC-NMR and HPLC-MS were successful in dereplicating the structures of several known compounds, as well as identifying the presence of new compounds in the crude extract of the bulbs of M. f ulginosa. This approach, in combination with the off-line phytochemical isolation, led to the identification of a total of 16 compounds representative of six different structure classes, including the identification of six new compounds (12−17). The isolated compounds were evaluated for antimicrobial activity, with only one compound (8) showing promising activity.

Table 3. 1H and 13C NMR Data (500 MHz, CDCl3) of Fuliginone (15) position

δC,a mult

1 2 3 3a 4 5

180.5, 149.5, 113.8, 128.4, 130.5, 127.2,

C C CH C CH CH

6 6a 7 8 9 9a 9b 1′ 2′ 3′ 4′ 5′ 6′

129.8, 132.0, 136.6, 126.8, 131.1, 127.5, 124.4, 142.9, 128.1, 127.8, 127.3, 127.8, 128.1,

CH C CH CH CH C C C CH CH CH CH CH

δH (J in Hz)

gCOSY

7.17, s

gHMBCAD

1D NOE

1, 2, 4, 9b

4, 2′, 6′

7.72, d (7.5) 7.59, dd (7.5, 8.0) 7.94, d (8.0)

5 4, 6

3, 6, 9b 3a, 6a

3

5

4, 7, 9b

5, 7

8.28, d (7.5) 7.81, t (7.5) 8.74, d (7.5)

8 7, 9 8

6, 9, 9b 6a, 9a 1, 7, 9b

6, 8

7.49, 7.39, 7.44, 7.39, 7.49,

3′ 2′

1′, 4′, 3′, 3′, 1′,

m m m m m

6′ 5′

6′ 5′ 5′ 4′ 2′

a

Carbon assignments based on HSQCAD and gHMBCAD NMR experiments.

unassigned. Insufficient quantities of these compounds precluded a specific rotation from being obtained. Biological Activity Studies. Of the compounds isolated from M. f ulginosa, rutin (4) possesses a range of properties including antioxidant,23,24 antihypertensive,25 and antiviral26 activities, while anigopreissin A (5) has been reported to prevent wrinkles that are caused by muscular contractions.27 Anigorufone (7) inhibits the growth of the casual agent of

Table 4. 1H and 13C NMR Data (500 MHz, CDCl3) for Fuliginol (16) and 3-Chlorofuliginol (17) fuliginol (16) position 1 2 3 3a 4 5 6 6a 7 8 9 9a 9b 1′ 2′ 3′ 4′ 5′ 6′ 2-OH 5-OH 6-OCH3 a

δC, mult 180.4, 148.8, 114.0, 127.6, 122.0, 147.8, 142.8, 127.0, 145.7, 131.1, 128.4, 124.9, 121.2, 141.7, 129.5, 127.3, 127.3, 127.3, 129.5,

C C CH C CH C C C C CH CH C C C CH CH CH CH CH

61.5, CH3

δH (J in Hz)

3-chlorofuliginol (17)

gCOSY

gHMBCAD

7.07, s

1, 2, 4, 9b

7.49, s

3, 3a, 5, 6, 9b

7.63, d (7.5) 8.61, d (7.5)

9 8

6a, 7, 9, 9a, 1′ 1, 7, 8, 9a, 9b

7.50, m 7.44, m 7.44, m 7.44, m 7.50, m NDa NDa 3.10, s

3′ 2′

7, 1′, 3′, 4′ 1′, 2′, 4′, 5′ 2′, 3′, 5′, 6′ 1′, 3′, 4′, 6′ 7, 1′, 4′, 5′

6′ 5′

6

δC, mult 178.2, 146.1, 121.3, 124.4, 120.7, 147.9, 143.7, 127.7, 146.2, 131.1, 129.0, 125.9, 120.7, 141.6, 129.3, 127.3, 127.3, 127.3, 129.3,

C C Cb Cb CH C C C C CH CH C C C CH CH CH CH CH

61.6, CH3

δH (J in Hz)

gCOSY

8.10, s

gHMBCAD

3, 3a, 5, 6, 9b

7.65, d (7.5) 8.62, d (7.5)

9 8

6a, 1′ 1, 7, 9b

7.50, m 7.45, m 7.45, m 7.45, m 7.50, m NDa NDa 3.14, s

3′ 2′

3′, 1′, 2′, 1′, 4′,

6′ 5′

4′ 2′ 6′ 6′ 5′

6

Signal not detected. bSignals interchangeable. E



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Table 5. Antimicrobial Activity of the Crude Extracts and Pure Compounds Obtained from M. fuliginosa, Together with Commercial Standard Antibiotic and Antifungal Compounds, Showing Zones of Inhibition (mm) microorganism a

CH2Cl2 extract of flowers MeOH extract of flowers CH2Cl2 extract of bulbs MeOH extract of bulbs mixture of hemofluorones A (1) and B (2) rutin (4) anigopreissin A (5) 2-phenylnaphthalic anhydride (6) anigorufone (7) compound 8 mixture of compounds 9 and 10 isosalipurposide (11) fuliginosin A (12) fuliginosin B (13) fuliginosone (14) fuliginone (15) mixture of fuliginol (16) and 3chlorofuliginol (17) ampicillin (antibiotic) carbendazim (antifungal)

b

concentration

E. coli ATCC 25922

S. aureus ATCC 25923

S. aureus MRSAc 344/2-32

P. aeruginosad ATCC 27853

S. pyogenese 345/1

C. albicansf ATCC 10231

25 mg/mL 50 mg/mL 50 mg/mL 50 mg/mL 1 mg/mL

NDg NDg NDg NDg NDg

1 NDg 1 1 NDg

NDg NDg NDg 2 NDg

3 NDg 2 1 1

1 NDg 3 4 NDg

NDg NDg 1 1 NDg

1639 μM 2211 μM 3649 μM 3675 μM 1724 μM 1 mg/mL 3030 μM 2762 μM 2450 μM 3875 μM 3905 μM 1 mg/mL

NDg NDg NDg NDg 1 NDg NDg NDg NDg NDg NDg NDg

NDg 2 NDg NDg NDg 1 NDg 1 NDg 2 NDg NDg

NDg NDg NDg NDg NDg NDg NDg NDg NDg NDg NDg NDg

NDg NDg 2 NDg 10 NDg 2 NDg NDg 1 NDg NDg

NDg 1 NDg NDg 1 1 NDg 1 NDg NDg 2 3

NDg NDg NDg NDg NDg NDg NDg NDg NDg NDg NDg NDg

2862 μM 5230 μM

NDg NTh

15 NTh

3 NTh

2 NTh

20 NTh

NTh NDg

a

Escherichia coli. bStaphylococcus aureus. cMethicillin-resistant Staphylococcus aureus. dPseudomonas aeruginosa. eStreptococcus pyogenes. fCandida albicans. gIndicates no zone of inhibition detected. hIndicates not tested.

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HP0921. HRMS was also carried out on a Waters Xevo QTof mass spectrometer, with an atmospheric solids analysis probe (ASAP) source (probe temperature set to 300 °C; source temperature set to 80 °C; sampling cone voltage of 20.0 V). Accurate mass was obtained by lockmass using leucine enkephalin as the reference compound. All analytical HPLC analyses and method development were performed on a Dionex P680 solvent delivery system equipped with a PDA100 UV detector (operated using Chromeleon software). Analytical HPLC analyses were carried out using either a gradient method (0−2 min 10% CH3CN/H2O; 14−24 min 75% CH3CN/H2O; 26−30 min 100% CH3CN; and 32−40 min 10% CH3CN/H2O or 0−3.5 min 30% CH3CN/H2O; 8.5−40 min 50% CH3CN/H2O) or an isocratic method (either 70, 50, 45, 40, 30, or 25% CH3CN/H2O) on an Alltech Alltima HP C18 (250 × 4.6) 5 μm column at a flow rate of 1.0 mL/min. Semipreparative HPLC was carried out on a Varian Prostar 210 solvent delivery system equipped with a Prostar 335 PDA detector (operated using Star Workstation software) using either a gradient method (0−2 min 10% CH3CN/H2O; 14−24 min 75% CH3CN/ H2O; 26−30 min 100% CH3CN; and 32−40 min 10% CH3CN/H2O or 0−3.5 min 30% CH3CN/H2O; 8.5−40 min 50% CH3CN/H2O) or an isocratic method (either 70, 50, 45, 40, 30, or 25% CH3CN/H2O) using an Alltech Alltima C18 (250 × 10) 5 μm column at a flow rate of 3.5 mL/min. Single-Crystal X-ray Diffraction. Crystals of fuliginosone (14), fuliginol (16), and 3-chlorofuliginol (17) were obtained by dissolving the compounds in CH2Cl2 and allowing the solvent to evaporate at room temperature. Crystallographic diffraction data were collected with an Oxford SuperNova diffractometer using Cu radiation (k = 1.541 84 A). Data were reduced using the CrysalisPRO software. The temperature of the data collection was maintained at 130 K, using an Oxford Cryostream cooling device. The structure was solved by direct methods and difference Fourier synthesis and was refined on F2 (SHELXL-97).42 A thermal ellipsoid plot was generated using the program ORTEP-343 integrated within the WINGX program suite.44 Biological Evaluation. For the biological evaluation procedure, refer to Brkljača and Urban.45

EXPERIMENTAL SECTION

General Experimental Procedures. All organic solvents used were analytical reagent (AR or GR), UV spectroscopic, or HPLC grade with Milli-Q water also being used. Optical rotations were carried out using a 1.5 mL cell on a Rudolph Research Analytical Autopol IV automatic polarimeter, set to the Na 589 nm wavelength. UV/vis spectra were recorded on an Agilent Cary 60 spectrophotometer, using EtOH. FTIR spectra were recorded as a film using NaCl disks on a PerkinElmer Spectrum One FTIR spectrometer. Uncorrected melting points were recorded on a Gallenkamp melting point apparatus. 1H (500 MHz), 13C (125 MHz), and 1D NOE spectra were acquired in CDCl3 and DMSO-d6 on a 500 MHz Agilent DD2 NMR spectrometer with referencing to solvent signals (δH 7.26; δC 77.0 and δH 2.50; δC 39.5 ppm, respectively). Two-dimensional NMR experiments recorded included gradient correlation spectroscopy (gCOSY), heteronuclear single-quantum correlation spectroscopy with adiabatic pulses (HSQCAD), and gradient heteronuclear multiple-bond spectroscopy with adiabatic pulses (gHMBCAD). Silica gel flash chromatography was carried out using Davisil LC35 Å silica gel (40−60 mesh) with a 20% stepwise solvent elution from 100% petroleum ether (60−80 °C) to 100% CH2Cl2 to 100% EtOAc and finally to 100% MeOH. C18 vacuum liquid chromatography (VLC) was carried out on silica gel 60 RP-18 (40−63 μm) using a 20% stepwise solvent elution from 100% H2O to 100% MeOH, and, finally, to 100% CH2Cl2 or a 20% stepwise elution from 30% CH3CN/H2O to 90% CH3CN/H2O and a final elution of 100% CH3CN. ESI mass spectra were obtained on a Micromass Platform II mass spectrometer equipped with an LC-10AD Shimadzu solvent delivery module (50% CH3CN/H2O at a flow rate of 0.2 mL/min) in both the positive and negative ionization modes using cone voltages between 20 and 30 V. HRESIMS was carried out on an Agilent 6200 Series TOF system (ESI operation conditions of 8 L/min N2, 325 °C drying gas temperature, and 3500 V capillary voltage) equipped with an Agilent 1200 Series LC solvent delivery module (100% MeOH at a flow rate of 0.3 mL/min) in either the positive or negative ionization modes. The instrument was calibrated using the “Agilent Tuning Mix” with purine as the reference compound and the Hewlett−Packard standard F



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Plant Material. Several specimens of the plant (M. f uliginosa) were purchased from Kuranga Native Nursery (Mount Evelyn, Victoria, Australia) and from Gardenworld Nursery (Braeside, Victoria, Australia) in January 2011, October 2012, and November 2012. The bulbs of the plants purchased in 2011 and 2012 were used in this study, while the flowers were harvested from the plants purchased in 2012. The bulbs and flowers were separated and then stored at −80 °C until extraction. Voucher specimens were designated with the code numbers 2011-01a (bulbs), 2011-02a (bulbs), 2012-01a (bulbs), and 2012_05a (flowers), respectively, and are deposited in the School of Applied Sciences (Discipline of Applied Chemistry), RMIT University. Phytochemical Profiling. Chemical profiling was carried out on the CH2Cl2-soluble extracts of the bulbs and flowers using HPLCNMR and HPLC-MS methodologies. Details of these analyses are provided in the General ExpErimental Procedures section and in Brkljača and Urban.45 The CH2Cl2 extract of the bulbs (157.9 mg) was dissolved in HPLC-NMR grade CH3CN (1580 μL) and filtered through a 0.45 PTFE membrane filter (Grace Davison Discovery Sciences) for HPLC-NMR analysis. Owing to the poor solubility of the CH2Cl2 crude extract of the flowers in CH3CN, no HPLC-NMR was conducted. HPLC-MS analyses were carried out on the CH2Cl2 crude extracts of both the bulbs and flowers. Extraction and Isolation of Flowers. The flowers of the plant (31 g, wet weight) were extracted with 3:1 MeOH/CH2Cl2 (2 L). The crude extract was decanted and concentrated under reduced pressure and sequentially solvent partitioned (triturated) into CH2Cl2- and MeOH-soluble extracts, respectively. A portion of the MeOH extract of the flowers (200 mg) was subjected to Sephadex LH-20 column chromatography (100% MeOH) to yield seven fractions. The fourth fraction was subjected to RP-HPLC (using a gradient of 0−3.5 min 30% CH3CN/H2O; 8.5−40 min 50% CH3CN/H2O) to yield isosalipurposide (11) (0.7 mg, 0.01%). Another portion of the MeOH extract of the flowers (244 mg) was subjected to Sephadex LH-20 column chromatography (100% MeOH) to yield six fractions. The fourth fraction yielded a mixture (13.6 mg, 0.2%) of hemofluorones A (1) and B (2). The second fraction was subjected to RP-HPLC (25% CH3CN/H2O) to yield rutin (4) (4.6 mg, 0.08%). The third fraction was subjected to reversed-phase HPLC (30% CH3CN/H2O) to yield the p-hydroxycinnamate of salipurposide (8) (0.4 mg, 0.007%), a mixture of methyl trans- and cis-p-coumarate (9 and 10) (0.1 mg, 0.002%), and isosalipurposide (11) (0.1 mg, 0.002%). Another portion of the MeOH extract (83.4 mg) was subjected to C18 VLC (20% stepwise elution from 30% CH3CN/H2O to 90% CH3CN/H2O and finally to 100% CH3CN) to yield 11 fractions. The first fraction (50% CH3CN/H2O) was subjected to Sephadex LH-20 column chromatography (100% MeOH) to yield rutin (4) (6.4 mg, 0.1%). The second VLC fraction (50% CH3CN/ H2O) was subjected to Sephadex LH-20 column chromatography (100% MeOH) to yield 11 fractions. Fractions 5−8 were combined to yield the p-hydroxycinnamate of salipurposide (8) (2.1 mg, 0.04%). The percentage yields are reported on the basis of the dry mass of the flowers extracted. Extraction and Isolation of Bulbs. The bulbs of the plant (77 g, wet weight) were extracted with 3:1 MeOH/CH2Cl2 (2 L). The crude extract was decanted and concentrated under reduced pressure and sequentially solvent partitioned (triturated) into CH2Cl2- and MeOHsoluble extracts, respectively. The CH2Cl2 extract was subjected to flash silica gel column chromatography (20% stepwise elution from petroleum ether (60−80 °C) to CH2Cl2 to EtOAc and, finally, to MeOH). The 100% CH2Cl2 fraction yielded anigorufone (7) (35.8 mg, 0.05%). The 80% CH2Cl2/EtOAc fraction (first eluting band) was subjected to RP-HPLC (70% CH3CN/H2O) to yield 2-phenylnaphthalic anhydride (6) (0.8 mg, 0.001%), anigorufone (7) (6.0 mg, 0.009%), and fuliginosone (14) (1.6 mg, 0.002%). The 80% CH2Cl2/ EtOAc fraction (second eluting band) was subjected to Sephadex LH20 column chromatography (100% MeOH) to yield four fractions. The third fraction was subjected to reversed-phase HPLC (70% CH 3CN/H2 O) to yield anigorufone (7) (4.5 mg, 0.007%), fuliginosone (14) (1.5 mg, 0.002%), 2-phenyl-1H-phenalen-1-one (15) (1.9 mg, 0.003%), and fuliginol (16) (6.5 mg, 0.01%). A portion

of the MeOH extract (150 mg) was subjected to RP-HPLC (50% CH3CN/H2O) to yield anigopreissin A (5) (12.2 mg, 0.02%) and fuliginosin A (12) (4.0 mg, 0.006%). Another portion of the MeOH extract (220 mg) was subjected to C18 VLC (20% stepwise elution from H2O to MeOH and finally to CH2Cl2) to yield 10 fractions. The 20% H2O/MeOH fraction was subjected to reversed-phase HPLC (50% CH3CN/H2O) to yield anigopreissin A (5) (17.1 mg, 0.03%) and fuliginosin A (12) (5.6 mg, 0.008%). One of the fractions from the HPLC purification was further subjected to RP-HPLC (50% CH3CN/ H2O) to yield fuliginosin A (12) (0.6 mg, 0.001%) and fuliginosin B (13) (0.7 mg, 0.001%). The percentage yields are reported on the basis of the dry mass of the bulbs extracted. On-line (HPLC-NMR and HPLC-MS) Characterization of Compounds. Compounds Derived from the Flowers of M. f ulginosa. Hemofluorone A (5,8,9-trihydroxy-1H-naphtho[2,1,8mna]xanthen-1-one) (1): tR = 3.74 min; UV (extracted from PDA) (30% CH3CN/D2O) λmax 572 nm; HPLC-MS m/z 317.0453 (calcd for C19H9O5, 317.0450). Hemofluorone B (5,8,9-trihydroxy-3H-naphtho[2,1,8-mna]xanthen3-one) (2): tR = 4.44 min; UV (extracted from PDA) (30% CH3CN/ D2O) λmax 551 nm; HPLC-MS m/z 317.0453 (calcd for C19H9O5, 317.0450). Rutin (4): tR = 2.45 min; UV (extracted from PDA) (30% CH3CN/ D2O) λmax 353 nm; HPLC-MS m/z 609.1453 (calcd for C27H29O16, 609.1456). p-Hydroxycinnamate of salipurposide (8): tR = 15.05 min; UV (extracted from PDA) (30% CH3CN/D2O) λmax 317, 368 nm; HPLCMS m/z 579.1503 (calcd for C30H27O12, 579.1503). Compounds Derived from the Bulbs of M. f ulginosa. Anigopreissin A (5-[4-hydroxy-2-(4-hydroxyphenyl)-6-{(1E)-2-(4hydroxyphenyl)ethenyl}-3-benzofuranyl]-1,3-benzenediol) (5): tR = 4.11 min; UV (extracted from PDA) (50% CH3CN/D2O) λmax 263, 303, 355 nm; HPLC-NMR WET1D NMR (500 MHz, 50% CH3CN/ D2O) obtained from stop-flow HPLC-NMR mode δ 7.82 (2H, d, J = 9.0 Hz, H-2a/H-6a*), 7.77 (2H, d, J = 9.0 Hz, H-3a/H-5a*), 7.59 (1H, d, J = 1.0 Hz, H-10b°), 7.52 (1H, d, J = 16.0 Hz, H-7b∧), 7.42 (1H, d, J = 16.0 Hz, H-8b∧), 7.22 (2H, d, J = 8.0 Hz, H-2b/H-6b#), 7.20 (1H, s, H-12a), 7.15 (2H, d, J = 8.0 Hz, H-3b/H-5b#), 6.83 (1H, d, J = 1.0 Hz, H-14b°), 6.78 (2H, s, H-10a/H-14a), *#∧°signals interchangeable; HPLC-MS m/z 451.1189 (calcd for C28H19O6, 451.1182). 2-Phenylnaphthalic anhydride (6): tR = 12.35 min; UV (extracted from PDA) (50% CH3CN/D2O) λmax 240, 323 nm; HPLC-NMR WET1D NMR (500 MHz, 50% CH3CN/D2O) obtained from stopflow HPLC-NMR mode δ 8.80 (2H, d, J = 8.5 Hz, H-4/H-5#), 8.52 (1H, d, J = 8.0 Hz, H-7#), 8.29 (1H, dd, J = 8.0, 8.5 Hz, H-6), 8.27 (1H, d, J = 8.5 Hz, H-3), 8.10 (2H, d, J = 7.5 Hz, H-2′/H-6′), 7.94 (2H, m, H-3′/H-5′), 7.92 (1H, m, H-4′), #signals interchangeable; HPLC-NMR 13C NMR (125 MHz, 50% CH3CN/D2O) obtained from stop-flow HPLC-NMR mode δ 134.0 (CH, C-4/C-5#), 131.6 (CH, C-3), 130.4 (CH, C-2′/C6′), 129.8 (CH, C-4′), 129.2 (CH, C3′/C-5′), 129.1 (CH, C-6), 123.1 (CH, C-7#), #signals interchangeable; HPLC-MS m/z 275.0700 (calcd for C18H11O3, 275.0708). Anigorufone (2-hydroxy-9-phenyl-1H-phenalen-1-one) (7): tR = 19.18 min; UV (extracted from PDA) (50% CH3CN/D2O) λmax 410 nm; HPLC-NMR WET1D NMR (500 MHz, 50% CH3CN/D2O) obtained from stop-flow HPLC-NMR mode δ 8.73 (1H, d, J = 8.0 Hz, H-7#), 8.44 (1H, d, J = 8.0 Hz, H-4*), 8.22 (1H, d, J = 7.0 Hz, H-6*), 8.06 (1H, dd, J = 7.0, 8.0 Hz, H-5), 7.97 (1H, d, J = 8.0 Hz, H-8#), 7.84 (2H, m, H-3′/H-5′), 7.82 (1H, m, H-4′), 7.75 (2H, d, J = 7.5 Hz, H-2′/H-6′), 7.59 (1H, s, H-3), *#signals interchangeable; HPLCNMR 13C NMR (125 MHz, 50% CH3CN/D2O) obtained from stopflow HPLC-NMR mode δ 136.5 (CH, C-7#), 132.0 (CH, C-8#), 131.8 (CH, C-6*), 130.8 (CH, C-4*), 129.1 (CH, C-3′/C-5′), 128.5 (CH, C-2′/C-6′), 128.2 (CH, C-4′), 128.0 (CH, C-5), 114.7 (CH, C-3), *#signals interchangeable; HPLC-MS m/z 271.0765 (calcd for C19H11O2, 271.0759). Fuliginosin A [3-(3,5-dihydroxyphenyl)-4-hydroxy-2-(4hydroxyphenyl)benzofuran-6-carbaldehyde] (12) and B [5-(6(dimethoxymethyl)-4-hydroxy-2-(4-hydroxyphenyl)benzofuran-3G



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19#/C-20), 69.7 (CH, C-18#), 60.9 (CH2, C-21), ND (C-11/C-15), *#signals interchangeable. Fuliginosin A [3-(3,5-dihydroxyphenyl)-4-hydroxy-2-(4hydroxyphenyl)benzofuran-6-carbaldehyde] (12): dark yellow oil; UV (EtOH) λmax (log ε) 285 (3.75), 324 (3.77), 367 (3.79) nm; IR νmax 3208, 2926, 1682, 1607, 1515, 1443, 1397, 1324, 1276 cm−1; 1H NMR (500 MHz, DMSO-d6) see Table 1; 13C NMR (125 MHz, DMSO-d6) see Table 1; HRESIMS m/z 361.0715 (calcd for C21H13O6, 361.0712). Fuliginosin B [5-(6-(dimethoxymethyl)-4-hydroxy-2-(4hydroxyphenyl)benzofuran-3-yl)benzene-1,3-diol] (13): brown oil; UV (EtOH) λmax (log ε) 303 (3.08), 313 (4.08), 369 (3.53) nm; IR νmax 3419, 2923, 2131, 1645 cm−1; 1H NMR (500 MHz, DMSO-d6) see Table 1; 13C NMR (125 MHz, DMSO-d6) see Table 1; HRESIMS m/z 407.1138 (calcd for C23H19O7, 407.1131). Fuliginosone (3-phenylacenaphthylene-1,2-dione) (14): bright yellow needles (CH2Cl2); mp 217−222 °C; C18H10O2, M = 258.26, T = 130.0(2) K, λ = 1.5418 Å, orthorhombic, space group Pbca, a = 8.4671(4) Å, b = 7.3151(4) Å, c = 39.179(2) Å, V = 2426.7(2) Å3, Z = 8, Dc = 1.414 Mg/m3 μ(Cu Kα) 0.736 mm−1, F(000) = 1072, crystal size 0.27 × 0.07 × 0.02 mm, 6910 reflections measured, 2188 independent reflections (Rint = 0.052), the final R was 0.0493 [I > 2σ(I)] and wR(F2) was 0.1255 (all data). Crystallographic data for 14 have been deposited at the Cambridge Crystallographic Data Centre (CCDC 1044045), 12 Union Road, Cambridge, CB2 1EZ, UK (www. ccdc.cam.ac.uk/data_request/cif); UV (EtOH) λmax (log ε) 244 (4.43), 324 (3.76) nm; IR νmax 1719, 1590 cm−1; 1H NMR (500 MHz, CDCl3) see Table 2; 13C NMR (125 MHz, CDCl3) see Table 2; HRESIMS m/z 259.0752 (calcd for C18H11O2, 259.0759). Fuliginone (2-phenyl-1H-phenalen-1-one) (15): brown, amorphous solid; UV (EtOH) λmax (log ε) 333 (3.63), 368 (3.65), 431 (3.59) nm; IR νmax 3350, 2925, 2854, 1622, 1574, 1510, 1491, 1409, 1274, 1214 cm−1; 1H NMR (500 MHz, CDCl3) see Table 3; 13C NMR (125 MHz, CDCl3) see Table 3; HRASAPMS m/z 257.0856 (calcd for C19H13O, 257.0966). Fuliginol (2,5-dihydroxy-6-methoxy-7-phenyl-1H-phenalen-1one) (16): cocrystal with 3-chlorofuliginol, orange needles (CHCl3); did not melt below 360 °C; 0.57(C20H12ClO4) 0.43(C20H14O4), M = 337.79, T = 130.0(2) K, λ = 1.5418 Å, triclinic, space group P1̅, a = 7.1864(15) Å, b = 10.074(2) Å, c = 10.803(3) Å, α = 89.17(2)°, β = 77.53(2)°, γ = 82.260(18)°, V = 756.6(3) Å3, Z = 2, Dc = 1.483 Mg/ m3, μ(Cu Kα) 1.732 mm−1, F(000) = 350, crystal size 0.14 × 0.04 × 0.02 mm, 3885 reflections measured, 2213 independent reflections (Rint = 0.057), the final R was 0.0704 [I > 2σ(I)] and wR(F2) was 0.200 (all data). Crystallographic data for 16 and 17 have been deposited at the Cambridge Crystallographic Data Centre (CCDC 1044044), 12 Union Road, Cambridge, CB2 1EZ, UK (www.ccdc.cam. ac.uk/data_request/cif); UV (EtOH) λmax (log ε) 275 (4.10), 375 (3.91), 463 (3.54) nm; IR νmax 3369, 1618, 1569, 1393, 1215 cm−1; 1H NMR (500 MHz, CDCl3) see Table 4; 13C NMR (125 MHz, CDCl3) see Table 4; HRESIMS m/z 317.0814 (calcd for C20H13O4, 317.0814). 3-Chlorofuliginol (3-chloro-2,5-dihydroxy-6-methoxy-7-phenyl1H-phenalen-1-one) (17): cocrystal data (see fuliginol (16)); 1H NMR (500 MHz, CDCl3) see Table 4; 13C NMR (125 MHz, CDCl3) see Table 4; HRESIMS m/z 351.0427 (calcd for C20H1235ClO4, 351.0424).

yl)benzene-1,3-diol] (13): tR = 3.28 min; UV (extracted from PDA) (50% CH3CN/D2O) λmax 238, 268, 322, 362 nm; HPLC-NMR WET1D NMR (500 MHz, 50% CH3CN/D2O) obtained from stopflow HPLC-NMR mode δ 10.25 (s), 8.01 (s), 7.82 (d, J = 8.5 Hz), 7.79 (s), 7.51 (s), 7.17 (d, J = 8.5 Hz), 7.02 (s), 6.77 (s), 6.74 (s), 4.18 (s), 4.15 (s). Fuliginosone (3-phenylacenaphthylene-1,2-dione) (14): tR = 11.31 min; UV (extracted from PDA) (50% CH3CN/D2O) λmax 240, 345 nm; HPLC-NMR WET1D NMR (500 MHz, 50% CH3CN/ D2O) obtained from stop-flow HPLC-NMR mode δ 9.02 (1H, d, J = 8.0 Hz, H-4*), 8.88 (1H, d, J = 8.0 Hz, H-5*), 8.83 (1H, d, J = 8.0 Hz, H-6*), 8.29 (1H, dd, J = 8.0, 8.0 Hz, H-7), 8.07 (1H, d, J = 8.0 Hz, H8*), 7.80−7.96 (5H, m, H-2′/H-3′/H-4′/H-5′/H-6′), *signals interchangeable; HPLC-MS m/z 259.0766 (calcd for C18H9O2, 259.0759). Off-line Characterization of Compounds. Hemofluorone A (5,8,9-trihydroxy-1H-naphtho[2,1,8-mna]xanthen-1-one) (1): purple, amorphous solid; all off-line NMR and MS data were identical to literature data;8 13C NMR (125 MHz, DMSO-d6) δ 181.8 (C, C-1), 158.1 (C, C-6a), 148.1 (C, C-9#), 146.6 (C, C-8#), 141.6 (C, C-5*), 140.0 (C, C-5a*), 139.3 (CH, C-3), 135.5 (C, C-10b), 131.4 (CH, C12), 126.9 (CH, C-2), 124.5 (C, C-12a), 122.6 (CH, C-4), 121.8 (C, C-3a), 121.5 (C, C-12b), 118.3 (C, C-12c), 113.6 (CH, C-11), 107.0 (C, C-10a), 105.4 (CH, C-10), 102.5 (CH, C-7), *#signals interchangable. Hemofluorone B (5,8,9-trihydroxy-3H-naphtho[2,1,8-mna]xanthen-3-one) (2): purple, amorphous solid; all off-line NMR and MS data were identical to literature data.3,8 Rutin: brown oil; all off-line NMR, MS, and specific rotation data were identical to literature data17 and compared to an authentic standard of rutin. Anigopreissin A (5-[4-hydroxy-2-(4-hydroxyphenyl)-6-{(1E)-2-(4hydroxyphenyl)ethenyl}-3-benzofuranyl]-1,3-benzenediol) (5): brown oil; all off-line NMR and MS data were identical to literature data.4 2-Phenylnaphthalic anhydride (6): yellow, amorphous solid; all off-line UV and MS data were identical to literature data;2 1H NMR (500 MHz, CDCl3) δ 8.71 (1H, dd, J = 7.5, 1.5 Hz, H-7), 8.34 (1H, dd (J = 8.5, 1.5 Hz, H-5), 8.29 (1H, d, J = 8.5 Hz, H-4), 7.85 (1H, dd, J = 8.5, 7.5 Hz, H-6), 7.69 (1H, d, J = 8.5 Hz, H-3), 7.42−7.57* (5H, m, H-2′, H-3′, H-4′, H-5′, H-6′); 13C NMR (125 MHz, CDCl3) δ 161.0 (C, C-9), 150.2 (C, C-2), 140.3 (C, C-1′), 135.4 (CH, C-5), 134.2 (CH, C-4), 133.7 (CH, C-7), 131.8 (CH, C-3), 131.2 (C, C-4a), 131.1 (C, C-8a), 128.2−128.4* (CH, C-2′, C-3′, C-4′, C-5′, C-6′), 127.1 (CH, C-6), 119.2 (C, C-8), 115.5 (C, C-1), ND (C, C-10), * indicates signals overlapped. Anigorufone (2-hydroxy-9-phenyl-1H-phenalen-1-one) (7): orange, amorphous soild; all off-line NMR and MS data were identical to literature data.5 p-Hydroxycinnamate of salipurposide (2,3-dihydro-7-hydroxy-2(4-hydroxyphenyl)-5-{[6-O-[3-(4-hydroxyphenyl)-1-oxo-2-propen-1yl]-β-D-glucopyranosyl]oxy}-4H-1-benzopyran-4-one) (8): light brown, amorphous solid; all off-line NMR and MS data were identical to literature data.18 Mixture of methyl trans-p-coumarate (9) and methyl cis-pcoumarate (10): amorphous, brown solid; trans to cis ratio was 1:0.17, respectively; all off-line NMR and MS data were identical to literature data.16,46,47 Isosalipurposide (11): yellow, amorphous solid; all off-line NMR and MS data were identical to literature data;15 1H NMR (500 MHz, CD3OD) δ 8.05 (1H, d, J = 15.5 Hz, H-8), 7.65 (1H, d, J = 15.5 Hz, H-7), 7.61 (2H, d, J = 9.0 Hz, H-2/H-6), 6.83 (2H, d, J = 9.0 Hz, H-3/ H-5), 6.17 (1H, brs, H-14*), 5.95 (1H, brs, H-12*), 5.15 (1H, d, J = 7.0 Hz, H-16), 3.92 (1H, d, J = 12.0 Hz, H-21a), 3.75 (1H, dd, J = 4.5, 12.0 Hz, H-21b), 3.55 (1H, m, H-17), 3.52 (1H, m, H-19#), 3.48 (1H, m, H-20), 3.46 (1H, m, H-18#), *#signals interchangeable; 13C NMR (125 MHz, CD3OD) δ 192.9 (C, C-9), 160.5 (C, C-13), 159.7 (C, C4), 142.3 (CH, C-7), 130.4 (CH, C-2/C-6), 127.1 (C, C-1), 124.8 (CH, C8), 115.5 (CH, C-3/C-5), 105.6 (C, C-10), 100.4 (CH, C-16), 97.6 (CH, C-12*), 95.1 (CH, C14*), 73.6 (CH, C-17), 77.2 (CH, C-

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S Supporting Information *

Supporting Information including the on-line HPLC-NMR and HPLC-MS analyses from the phytochemical profiling together with the off-line NMR and high-resolution MS data associated with this study. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jnatprod.5b00161. H



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AUTHOR INFORMATION

Corresponding Author

*Tel: +61-3-9925-3376. Fax: +61-3-9925-3747. E-mail: sylvia. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Marine and Terrestrial Natural Product (MATNAP) research group would like to thank Mrs. N. Thurbon (School of Applied Sciences (Discipline of Biotechnology and Biological Sciences), Science Engineering and Health, RMIT University) for providing access to the microorganisms to conduct the antimicrobial assays and for her invaluable technical support; Ms. S. Duck (School of Chemistry, Faculty of Science, Monash University) for conducting the HRMS analyses along with access to the HPLC-MS instrument; and Dr. J. Niere for NMR discussions and guidance. R.B. would also like to acknowledge his Australian Postgraduate Award (APA) scholarship that has supported his Ph.D. studies.

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